Expression and characterization of an N-truncated form of the NifA protein of Azospirillum brasilense

Azospirillum brasilense is a nitrogen-fixing bacterium associated with important agricultural crops such as rice, wheat and maize. The expression of genes responsible for nitrogen fixation (nif genes) in this bacterium is dependent on the transcriptional activator NifA. This protein contains three structural domains: the N-terminal domain is responsible for the negative control by fixed nitrogen; the central domain interacts with the RNA polymerase σ54 co-factor and the C-terminal domain is involved in DNA binding. The central and C-terminal domains are linked by the interdomain linker (IDL). A conserved four-cysteine motif encompassing the end of the central domain and the IDL is probably involved in the oxygen-sensitivity of NifA. In the present study, we have expressed, purified and characterized an N-truncated form of A. brasilense NifA. The protein expression was carried out in Escherichia coli and the N-truncated NifA protein was purified by chromatography using an affinity metal-chelating resin followed by a heparin-bound resin. Protein homogeneity was determined by densitometric analysis. The N-truncated protein activated in vivo nifH::lacZ transcription regardless of fixed nitrogen concentration (absence or presence of 20 mM NH4Cl) but only under low oxygen levels. On the other hand, the aerobically purified N-truncated NifA protein bound to the nifB promoter, as demonstrated by an electrophoretic mobility shift assay, implying that DNA-binding activity is not strictly controlled by oxygen levels. Our data show that, while the N-truncated NifA is inactive in vivo under aerobic conditions, it still retains DNA-binding activity, suggesting that the oxidized form of NifA bound to DNA is not competent to activate transcription.

Efecto de la biofertilización y los biorreguladores en la germinación y el crecimiento de carica papaya L / Effect of biofertilization and bioregulators on germination and growth of carica papaya L

Con el objetivo de incrementar y acelerar el proceso de germinación de las semillas y obtener una alta producción y homogeneidad de plántulas de Carica papaya variedad Maradol en vivero, se evaluó el efecto de tres biofertilizantes aplicados solos o en combinación (Azotobacter chroococcum, Azospirillum brasilense y Glomus intraradices), y un biorregulador del crecimiento vegetal, el ácido giberélico (AG3), en la germinación y el crecimiento vegetal. Se realizó un experimento bajo un diseño completamente al azar con ocho tratamientos y tres repeticiones. A las semillas se les aplicó un pretratamiento germinativo con alternancia de temperatura para superar la dormancia. Los tratamientos simples con A. chroococcum y A. brasilense, incrementaron el porcentaje de germinación a 90,28 y 88,89% respectivamente. Además, con la aplicación de los biofertilizantes y el AG3, la velocidad de germinación se incrementó y el tiempo medio de germinación se redujo. La doble aplicación en semillas y foliar de los biofertilizantes y el AG3 en plántulas mejoró el crecimiento vegetal. La población de A. chroococcum fue mayor cuando se inoculó en combinación con G. intraradices. La prevalencia de colonización de las plántulas inoculadas con G. intraradices varió de 18,53 a 26,67%, con el mayor valor registrado para el tratamiento combinado con A. brasilense. Finalmente, aplicando esta metodología se logró acelerar la germinación, obteniéndose una mayor homogeneidad en la emergencia de las plántulas, disminuyendo así el tiempo de permanencia en el vivero.

In order to increase and accelerate the process of seed germination and obtain a high yield and homogeneity of papaya seedlings cv. Maradol in nurseries, we evaluated the effect of three biofertilizers applied single or in combination (Azotobacter chroococcum, Azospirillum brasilense and Glomus intraradices) and a plant growth bioregulator, the gibberellic acid 3 (AG3), on the germination and subsequent growth of papaya seedlings. An experimental design completely random with eight treatments and three replications were used. The application of a pre-germinal treatment with alternating temperature had to be applied to seeds to overcome dormancy. Single biofertilization with A. chroococcum and A. brasilense, promoted the germination percentage 90.28 y 88.89% respectively. Germination rate could be enhanced and the mean germination time was reduced with the application of biofertilizer and AG3. Both applications on seeds and leaves of biofertilizers and AG3, had a positive effect on plant growth. The population of A. chroococcum was higher in the combined inoculation with G. intraradices. The prevalence of colonization of plants inoculated with G. intraradices ranged from 18.53 to 26.67%, with the greatest values recorded for the treatment involving combined inoculation with A. brasilense. Finally, with the application of this methodology the seed germination rate was improved, as well as the uniformity of seedlings emergence...

Caracterização parcial de um pigmento produzido por Azospirillum lipoferum / Partial characterization of a brown pigment produced by Azospirillum lipoferum

Dezenove cepas de Azospirillum lipoferum e quarenta e uma de Azospirillum brasilense foram examinadas para a produção de pigmento em três meios suplementados com L-tirosina. Doze ceps de A. lipoferum apresentaram essa capacidade no meio NFb líquido enriquecido com L-tirosina. Uma pigmentação marrom intensa foi produzida apenas por A. lipoferum USA5a. Esta cepa foi utilizada como a produtora do pigmento marrom neste estudo. Quando L-tirosina foi substituída por 21 aminoácidos e D-L ácido málico por outras fontes de carbono, verificou-se que o pigmento foi também intensamente produzido com L e DL triptofano e lactato de sódio. A presença de ferro foi essencial para a ocorrência da pigmentação. A cor marrom apresentada pelo pigmento sugere tratar-se de melanina. Embora tenha se comportado como esta substância em alguns testes de caracterização de malanina, o pigmento produzido por A. lipoferum USA5a apresenta características distintas. Além disso, nenhuma atividade da tirosinase foi encontrada nos extratos de células examinados com L-tirosina e L-DOPA. Este pigmento marron parece estar relacionado com o catabolismo da tirosina.